SUMMARY The goal of this proposal is to use Organs-on-Chips (Organ Chips) to develop clinically relevant in vitro models of influenza infection in humans that can be used to test efficacy of candidate therapeutics, explore variation in responses in different patient populations, and potentially develop anti-influenza drugs that target the host response to infection, rather than the virus itself. Our Organ Chips are 2-channel microfluidic culture devices that are lined by human organ-specific tissue cells and vascular endothelium grown in parallel microchannels separated by a porous extracellular matrix-coated membrane. We have previously created Lung Alveolus Chips as well as Small Airway Chips lined by bronchiolar epithelial cells from either normal donors or diseased patients, such as individuals with chronic obstructive pulmonary disease (COPD), and we showed that they faithfully recapitulate human pathophysiology observed in vivo, including lung inflammation and pulmonary edema In addition, we have created human Liver Chips that metabolize drugs in vitro, and engineered an instrument for automated culture and fluidic coupling of up to 10 human organ chips for up to 4 weeks, which can be used to link different Organ Chips in a physiological way. Importantly, in preliminary studies, we successfully infected these bronchiolar epithelium with H1N1 influenza A virus (IAV), identified molecular mediators of the host response to infection, and discovered a potential new antiviral therapeutic that targets these mediators. In the UG3 phase of this project, we will demonstrate that Lung Airway and Alveolus Chips lined by primary cells isolated from human healthy donors or COPD patients can be used to model clinical features of IAV infection and related lung disorders previously observed in human patients, including viral replication and shedding, release of characteristic inflammatory cytokines, recruitment of circulating immune cells, and pulmonary edema, all of which we will measured non-invasively. During the UH3 phase, we will conduct preclinical efficacy testing of existing antiviral drugs and use multi-omics analysis and bioinformatics approaches to define translatable biomarkers and identify new potential molecular targets. We also will leverage these insights to discover new potential therapeutics that target the host response to infection, rather than the virus itself. Our UG3 Specific Aims are 1) to develop models of influenza infection in human Lung Airway and Alveolus Chips lined by cells from healthy donors and COPD patients that recapitulate in vivo disease responses, and 2) to develop an integrated model for influenza drug testing by fluidically linking Lung Airway, Lung Alveolus Chips, and Liver Chips via their vascular channels. Our UH3 Aims include: 1) to use the integrated Organ Chip influenza model to measure efficacy and safety of known antiviral therapeutics, 2) to validate translatable biomarkers for influenza infection and therapeutic responses identified using the Organ Chip model by comparison with clinical measures in humans, and 3) to use the integrated Organ Chip influenza model to identify new antiviral therapeutics that target host responses to infection.